Am. J. Trop. Med. Hyg., 93(2), 2015, pp. 350–355 doi:10.4269/ajtmh.15-0236 Copyright © 2015 by The American Society of Tropical Medicine and Hygiene

Case Report: Thirty-Seven Human Cases of from Ethiopia and South Sudan Caused by Spp.

Mark L. Eberhard,* Elizabeth A. Thiele, Gole E. Yembo, Makoy S. Yibi, Vitaliano A. Cama, and Ernesto Ruiz-Tiben Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia; Ethiopia Eradication Program, Federal Ministry of Health, Addis Ababa, Ethiopia; South Sudan Guinea Worm Eradication Program, Ministry of Health, Juba, Republic of South Sudan; The Carter Center, Atlanta, Georgia

Abstract. Thirty-seven unusual specimens, three from Ethiopia and 34 from South Sudan, were submitted since 2012 for further identification by the Ethiopian Dracunculiasis Eradication Program (EDEP) and the South Sudan Guinea Worm Eradication Program (SSGWEP), respectively. Although the majority of specimens emerged from sores or breaks in the skin, there was concern that they did not represent bona fide cases of Dracunculus medinensis and that they needed detailed examination and identification as provided by the World Health Organization Collaborating Center (WHO CC) at Centers for Disease Control and Prevention (CDC). All 37 specimens were identified on microscopic study as larval tapeworms of the spargana type, and DNA sequence analysis of seven confirmed the identification of Spirometra sp. Age of cases ranged between 7 and 70 years (mean 25 years); 21 (57%) patients were male and 16 were female. The presence of spargana in open skin lesions is somewhat atypical, but does confirm the fact that populations living in these remote areas are either ingesting infected in unsafe drinking water or, more likely, eating poorly cooked paratenic hosts harboring the parasite.

INTRODUCTION gated within 24 hours. Any emergent worms are collected and preserved in alcohol and sent to CDC for subsequent Sparganosis, or human infection with the larval plerocer- examination. Thirty-seven of those specimens, three from coid stage of members of the Spirometra (: Ethiopia and 34 from South Sudan, were microscopically ), is a rather common infection in south- 1–4 identified as spargana; 36 were collected through the national east (SE) Asia, but is much less frequently reported in programs to eliminate GWD and one from a surgical proce- other areas of the world, despite being a common infection dure for varicose vein. The three specimens submitted from in cats and dogs throughout much of the world. In Africa, Ethiopia over that time were out of a total of 21 specimens, there are limited reports of human cases dating back to 1907 and for South Sudan, the 34 spargana were out of a total of when the first case was noted, and since then there have 161 submitted samples. The cases ranged from 7 to 70 years been sporadic reports and records of human cases although of age, with a median age of 25 years. Twenty-one (57%) of these same authors have speculated that human sparganosis 5–7 the cases were male, 16 were female. For the 34 cases from is a relatively common infection, at least in east Africa. South Sudan, a map showing the location of those cases is As the end of the campaign to eradicate Guinea worm dis- provided (Figure 1). Those 34 cases emerged in 17 different ease (GWD) progresses and the number of endemic countries villages, and in those with multiple cases, the number of cases and cases decreases, there is increased effort to detect and per each individual village were 2, 2, 4, 5, and 9. Specimens contain each case. Concurrent with this is the need to con- were taken from various locations on the body (Figure 2), firm that each case is accurately identified as Dracunculus including thigh, lower leg, ankle, foot, knee, and penis and medinensis so that appropriate actions at the country pro- measured between 0.5 and 30 cm long, were whitish-tan in gram level can be undertaken. However, not all specimens 8,9 color, and often demonstrated a thicker anterior end and a recovered from or emerging via the skin are Dracunculus. very thin posterior end (Figure 3). Under low-power micro- Up to now, the most common non-Dracunculus recovered by scopic evaluation, rudimentary bothria (cephalic grooves) typical national programs and submitted to the World Health Organi- of pseudophyllidian tapeworms was clearly evident on some zation Collaborating Center (WHO CC) at Centers for Dis- specimens. However, not all specimens contained the anterior ease Control and Prevention (CDC) has been Onchocerca end, and ranged from short to very long ribbon-like pieces of volvulus. However, over the past 3 years, an increasing num- worm (Figure 4). In those specimens, identification was a bit ber of spargana have been submitted and this report briefly more challenging, but the presence of calcareous corpuscles details 37 such cases, two each from 2012 and 2013, and 33 (Figure 5) in the somatic matrix was particularly helpful in from 2014. establishing that the organism was a larval tapeworm. Nine of the submitted specimens were subjected to DNA CASE SERIES REPORT sequencing to further verify the morphological identification. In brief, whole genomic DNA was extracted using a Gentra As part of routine, ongoing case detection and containment Puregene Tissue Kit (Qiagen, Frederick, MD) and used for activities in remaining endemic countries, all suspect cases amplification of the nuclear 18S rDNA and mitochondrial (and rumors) of dracunculiasis are supposed to be investi- cytochrome oxidase 1 (CO1) genes. The 18S rDNA amplifica- tion was performed with general primers commonly used in molecular verification of D. medinensis to confirm that *Address correspondence to Mark L. Eberhard, Division of 10 Parasitic Diseases and Malaria, Centers for Disease Control and samples were not morphologically atypical guinea worms. In Prevention, 1600 Clifton Road, Atlanta, GA 30333. E-mail: mle1@ all nine cases, 18S rDNA reactions were negative, supporting cdc.gov the original diagnosis that specimens were not D. medinensis. 350 HUMAN SPARGANOSIS IN EAST AFRICA 351

FIGURE 1. Map of South Sudan indicating the location of 17 villages or village clusters where 34 cases of human sparganosis were detected during 2013–2014.

CO1 sequences were generated with a custom set of primers the 983 bp of shared CO1 sequence and were polymorphic at designed to serve as a general primer set for diphyllobothriid 10 single nucleotide sites (average pairwise polymorphism: CO1 amplification. The forward primer sCO1 F (5′-GAT 2.86 nucleotides, range: 0–8 nucleotides). BLAST queries of AGHMRGGGTGTTGATYTT-3′) and reverse primer sCO1 the National Center for Biotechnology Information (NCBI) R(5′-CCARATAGCATGATGCAAA-3′; modified from the nucleotide database indicated that, of available sequences, the diphyllobothriid Cox1Reverse primer of Wicht and others11) African spargana described here had highest similarity to were used for polymerase chain reaction (PCR) amplification. Spirometra erinaceieuropaei (89–90% identity over 983 bp). Sig- For cycle sequencing of PCR product, two internal primers nificant homologies were also returned for the diphyllobothriid (sCO1 intF: 5′-TGTTTACDGTRGGDTTRGAYG-3′ and genera Sparganum, ,andDiplogonoporus sCO1 intR: 5′-CAAGCRTAMCCBGACTCRT-3′)wereused and the cyclophyllid genus Echinococcus,with≥ 91% coverage in addition to the PCR primers so as to ensure complete of African spargana sequences. bidirectional coverage. Genetic distances (Kimura 2-parameter model, bootstrap Seven of the nine specimens were successfully amplified in N = 1,000) and phylogenetic trees (maximum-likelihood using 50 μL reactions comprising 1 μL of whole genomic DNA, 1X a GTR+G+I substitution model) were calculated and inferred, Platinum® Blue PCR Supermix (Invitrogen, New York, NY), respectively, in MEGA v6.012 (freely available at http://www and 0.4 μM of each CO1 primer. Cleaned PCR product .megasoftware.net) to further investigate the taxonomic relation- (StrataPrep PCR Purification Kit [Agilent Technologies, Foster ship among the African spargana, Spirometra erinaceieuropaei, City, CA]) was sequenced using the BigDye® Terminator v3.1 and other diphyllobothriid and Echinococcus species known or cycle sequencing kit (Applied Biosystems, New York, NY) previously reported to occur in humans. Representative CO1 andanalyzedona3130xl Genetic Analyzer (Applied Bio- gene sequences of Spirometra erinaceieuropaei (AB015754), systems). Electropherograms were visually inspected and the Sparganum proliferum (AB015753), Diphyllobothrium ditremum sequences trimmed and assembled with ChromasPro v1.7.4 (FM209182), D. nihonkaiense (AB268585), D. latum (AB269325), (Technelysium, South Brisbane, Australia). High-quality par- D. dendriticum (KC812048), Diplogonoporus balaenopterae tial CO1 gene sequences of 996–1,027 base pairs (bp) were (AB425839), Dg. grandis (AB425840), Echinococcus canadensis generated for the seven successfully amplified specimens and (AB745463), E. multilocularis (AB510023), and E. granulosus were submitted to GenBank (accession numbers KM248530- (GQ168812) were obtained from GenBank, with the cestodarian 248536). Overall, the seven specimens had 99% identity over species Gyrocotyle urna (JQ268546) serving as the out-group. 352 EBERHARD AND OTHERS

FIGURE 2. Clinical presentation of a case of sparganosis illustrating the progression of the emergence of the spargana. (A) Initial lesion with minute white tip of worm. (B) Protrusion of larger portion of larva. (C) Mass of “hanging” worm. (D) Lesion after final emergence of larva. These images show the remarkable similarity to the appearance of emergence of Guinea worm.

Homology of all alignments was supported with amino acid the inferred phylogenetic treegroupedthesevenAfrican translation before analysis. Average genetic divergence spargana into a distinct clade, sister to Spirometra erinaceieuropaei within the African spargana was quite low (d = 0.004 ± 0.001) within the larger diphyllobothriid clade (Figure 6). compared with divergence between African spargana and Spirometra erinaceieuropaei (d = 0.107 ± 0.012) and all other interspecies and intergenus comparisons (d ≥ 0.075). Similarly, DISCUSSION ThelifecycleoftheDiphyllobothriidea, including Spirometra, differs considerably from that of the and typi- cally includes a first and second intermediate host. On reaching water, the egg hatches and releases a ciliated coracidium that is ingested by a , in which the procercoid stage develops. When the infected copepod is ingested by an appropriate second intermediate host, the parasite develops to the plero- cercoid stage (= spargana). Birds, reptiles, and amphibians serve as usual second intermediate hosts but many other ani- mals can become infected. If these hosts are eaten by other than the normal definitive host, the parasite can infect that , but undergoes no further development. Some hosts may serve either as a second intermediate or a paratenic host. When one of these hosts carrying the spargana is eaten by the normal definitive host, a canine or feline depending on the parasite species, the parasite develops into the adult tape- worm in the small intestine. This entire cycle from egg laying to infection in a new definitive host can occur in approxi- mately 3 months, about a quarter of the time required for

FIGURE 3. Gross image of entire spargana. Higher magnification completion of the Guinea worm life cycle. In contrast to showing rudimentary bothrium is shown in inset. Diphyllobothrium, there are some questions about whether HUMAN SPARGANOSIS IN EAST AFRICA 353

FIGURE 5. High power image of squash prep of piece of spargana showing the spherical calcareous corpuscles.

tive role that each contributes to the overall infection rate is not known. Despite the dearth of reports of sparganosis in humans from Africa, the occurrence of adult Spirometra seems to be both common and widespread, at least in east Africa. Domestic dogs and cats are typical hosts, although Mueller13 felt that in Africa, dogs were the preferred host. In addition to domestic animals, wild carnivores are frequent hosts. Hyenas in Kenya and Zambia were noted to have a high rate of infection, with 52/70 (74%) and 2/9 (22%), respectively, of animals examined positive for eggs in the feces,14,15 and 63% of 112 lions and 87% of 15 lions examined in Tanzania and Zambia, respectively, found to be infected as determined by – finding eggs in the feces.15 17 Spirometra was the most com- monly found parasite in the examined lions and second most common parasite in the hyenas. The parasite in the hyenas is believed to be Spirometra pretoriensis and that from the lions Spirometra theileri, although some adult worms from lions could not be distinguished from Spirometra pretoriensis, suggesting that both species may occur in lions. Wild herbi- vores such as antelope, buffalo, zebra, and warthog have been found to be commonly infected with the pleroceroid stage,5,16 and when these animals fall prey to carnivores such as hyenas and lions, the life cycle is completed.18 Opuni and Muller19 were able to experimentally infect dogs but not cats with spargana recovered from warthogs in Tanzania. They were also unable to infect various amphibians or reptiles with oral dosing of infected copepods, which calls into ques- tion what role typical second intermediate and paratenic hosts such as amphibians and reptiles may play in the life cycle of

FIGURE 4. Gross image of three different pieces of spargana certain of these African species. Further, it should be noted that were submitted illustrating the range in size and shape of that at least in east Africa not all recovered tapeworm larvae recovered material. of the spargana type are actually Spirometra. Nelson and others5 found that a number of larval tapeworms recovered from various animals were actually tetrathyridia, the larval fishplayaroleintheSpirometra life cycle and can serve as stage of Mesocestoides, and not spargana. Because they second intermediate hosts. Humans serve as second intermedi- resemble each other superficially, some reports of spargana ate or paratenic hosts, becoming infected with the plerocercoid in animals likely represent tetrathyridia. However, larval (spargana) by ingestion of poorly cooked intermediate or Mesocestoides are not thought to infect humans. paratenic hosts, by using the flesh of an infected second inter- Baboons, vervets, and other monkeys are also infected in mediate or paratenic host as poultice on an open wound or east Africa.20,21 The species infecting monkeys has not been the eye, or by ingestion of infected copepods.1,2 The rela- established but is thought to be either Spirometra theileri, 354 EBERHARD AND OTHERS

FIGURE 6. Phylogenetic relationship of seven east African spargana to diphyllobothriid and cyclophyllidean cestodes known or previously reported to infect humans. The tree was inferred with maximum likelihood analysis of partial CO1 gene sequences (860 base pairs [bp]), using the cestodarian species Gyrocotyle urna as the out-group. To focus on the association of the African spargana with other known agents of sparganosis, branches for the Diphyllobothrium, Diplogonoporus, and Echinococcus genera have been collapsed. Branch lengths are shown in number of substitutions per site as indicated by the scale bar, and nodal support is indicated where > 70 (bootstrap, N = 1,000). National Center for Biotechnology Information (NCBI) accession numbers of sequences generated in this study are shown in parentheses.

Spirometra mansonoides,orSpirometra proliferum, although hosts, very much similar to what was proposed for the recent it could well represent some other species. The range of species peculiar epidemiology of GWD in Chad.26 The finding of of Spirometra that occur in Africa is unclear, and given the a number of human cases of sparganosis substantiates the wide host susceptibility observed in the second intermediate potential for such an unusual life cycle for guinea worm to be and paratenic hosts, additional work is clearly needed to sort happening, and suggests that some people in rural and remote out not only the , but also the differences in the life areas, at least in South Sudan and Ethiopia, are drinking unpro- cycle between those species that have different definitive hosts. tected water, and, more likely, eating poorly cooked animals that When cases of sparganosis are found, microscopy can be serve as paratenic hosts, such as frogs, snakes, or other animals. the first line of identification. However, further identification Finally, the use of poultices and other natural remedies remains beyond that generic determination requires molecular analysis common in remote populations and cannot be discounted as as was the case in this study. The literature to date indicates a potential means of infection for some of these cases. that the CO1 gene provides optimal genetic variation for phy- logenetic distinction and species-level identification of the Received March 25, 2015. Accepted for publication April 8, 2015. 22–25 Diphyllobthriidea. As such, CO1 sequencing was our chosen Published online June 8, 2015. method for molecular identification of the unknown spargana Acknowledgments: We thank Julie A. Krause, Technical Advisor, recovered in west Africa. CO1 sequences of the Ethiopian and The Carter Center, for sharing the photographs used in Figure 2. South Sudanese spargana described here exhibited highest similarity to those of Spirometra erinaceieuropaei largely origi- Disclaimer: The findings and conclusions herein are those of the authors and do not necessarily represent the official position of the nating from SE Asia. However, both the estimates of sequence Centers for Disease Control and Prevention. divergence and inferred phylogeny suggest that the African Authors’ addresses: Mark L. Eberhard, Elizabeth A. Thiele, and spargana reported here are a single sister species to Spirometra Vitaliano A. Cama, Division of Parasitic Diseases and Malaria, Cen- erinaceieuropaei (Figure 5), though no further designation than ters for Disease Control and Prevention, Atlanta, GA, E-mails: Spirometra sp. can be applied at this time. Whether the human [email protected], [email protected], and [email protected]. Gole E. Yembo, infections documented in this report represent or are related Federal Ministry of Health, Addis Ababa, Ethiopia, E-mail: gole_ to species of Spirometra known to commonly infect large carni- [email protected]. Makoy S. Yibi, Ministry of Health, Juba, Republic of South Sudan, E-mail: [email protected]. Ernesto Ruiz- vores in east Africa (e.g., Spirometra theileri or Spirometra Tiben, The Carter Center, Atlanta, GA, E-mail: [email protected]. pretoriensis)isunclearandwilllikelynotbefullyresolveduntil This is an open-access article distributed under the terms of the further molecular work is done on those species. Creative Commons Attribution License, which permits unrestricted It is noteworthy that the life cycle of Spirometra utilizes use, distribution, and reproduction in any medium, provided the copepods as first intermediate host and various paratenic original author and source are credited. HUMAN SPARGANOSIS IN EAST AFRICA 355

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